solation and quantification of flavonoids from ethanol extract of Costus igneushizome (CiREE) and impact of CiREE on hypoglycaemic, electron microscopictudies of pancreas in streptozotocin (STZ)-induced diabetic rats
Department of Biotechnology, Periyar Maniammai University, Vallam, 613403 Tamilnadu, IndiaDry land Agricultural Research Station, Tamil Nadu Agricultural University, 630102 Chettinad, India
a r t i c l e i n f o
rticle history:eceived 9 January 2013ccepted 21 January 2013
eywords:ostus igneusntidiabeticaempferol
a b s t r a c t
The aim of the study was to isolate and quantify flavonoids from ethanol extract of Costus igneus rhi-zome (CiREE) and to find the impact of CiREE on hypoglycaemic effect, electron microscopical study ofpancreas in streptozotocin (STZ)-induced diabetic rats. Quercetin (Rf- 0.72, 0.794%) and kaempferol (Rf-0.35, 4.2%) were quantified by HPTLC using solvent ratio of Toluene:Ethyl acetate:Acetic acid:Methanol(2:7:0.25:0.25) and Toluene:Ethyl acetate:Methanol:Formic acid (6:3:0.2:0.4) respectively. Quercetinderivative compound was characterized by H1, C13 NMR. The CiREE at a dose of 100, 200 mg/kg wereorally administered as a single dose per day to diabetes-induced rats for a period of 30 days. The effect ofCiREE on blood glucose, plasma insulin, liver glycogen, HbA1c and serum lipid profile (Total cholesterol[TC], Triglyceride [TG], Very low density lipoprotein [VLDL], Low density lipoprotein [LDL], High densitylipoprotein [HDL]) were measured, histopathological and electron microscopical studies of pancreas weredone in normal and diabetic rats. The results showed that fasting rat’s blood glucose, serum TC, TG, LDL,VLDL levels were significantly (P < 0.05) increased whereas serum HDL, glycogen and insulin level were
significantly (P < 0.05) decreased in the diabetic rats, after treatment with CiREE these levels were reverseback to normal. The dosage of 200 mg/kg is more effective than that of 100 mg/kg. Histopathologicaland Electron microscopical study of pancreas revealed that number of �-cells and insulin granules areincreased in diabetes-induced rats after treatment with CiREE for 30 days. Our investigation thus showsthat CiREE has potent antidiabetic and hypolipidemic effects in STZ-induced diabetic rats and these effectswere much comparable to that of the standard reference drug glibenclamide.
. Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized byyperglycemia resulting from faults in insulin secretion, insulinction, or both [1]. Diabetes is becoming the third killer diseasef mankind, after cancer and cardiovascular disease, because of itsigh prevalence, morbidity and mortality [2]. The presence of DMonfers increased risk of many devastating complications such asardio vascular disease, peripheral vascular complications such asoronary artery disease, stroke, neuropathy, renal failure, retinopa-
hy and blindness. Insulin and various types of hypoglycemic agentsuch as biguanides and sulfonylureas are available for the treatmentf diabetes. The main disadvantages of the currently available drugs
are that they have to be taken throughout the life characterizedwith side effects [3]. But most of traditional treatments have beenrecommended in the complementary and alternative medicine forthe treatment of diabetic mellitus. Based on the WHO recom-mendation antihyperglycemic compounds of plant origin used intraditional medicine are of more important and which are less toxicand free from side effects [4].
The mechanism of the herbals used to treat diabetes has not yetbeen defined. It has been attributed that the hypoglycemic effect ofthese plants is due to their ability to restore or stimulate the func-tion of pancreatic tissues or �-cells by causing an increase in insulinoutput or inhibit the intestinal absorption of glucose or to by thefacilitation of metabolites in insulin dependent biochemical pro-
cess. Hence treatment with herbal drugs has an effect on protectingor regenerating the �-cells and stimulate the insulin secretion tomaintain the blood glucose level. Costus igneus also known as fierycostus or spiral flag or insulin plant belongs to the costaceae family,
ontains a range of phytochemicals viz flavonoids, alkaloids, ter-enoids and it was traditionally used in India to control diabetesnd experimental diabetic rats [5–11]. The present study is con-ucted to investigate the antidiabetic compounds, antidiabetic andypolipidemic effect of C. igneus ethanol rhizome extract (CiREE) intreptozotocin (STZ)-induced diabetic male albino rats.
. Materials and methods
.1. Extraction and active compound isolation
.1.1. Plant materialsThe fresh C. igneus rhizome was collected from Periyar Mani-
mmai University nursery in the month of April (2008). Plant wasdentified, confirmed and authenticated by Rapinant Herbarium, St.oseph’s College, Trichy, Tamil Nadu, South India.
.1.2. Drugs and chemicalsSTZ was purchased from Himedia laboratories Pvt. Ltd, Mumbai,
ndia. Standard antidiabetic drug glibenclamide was obtained fromanbaxy Research Laboratories, Gurgaon, India. Analytical gradehemicals, including various organic solvents (petroleum ether,exane, ethanol and methanol) from merk and loba chemicals,
ndia, were used for the extraction and to study the phytochemicalonstituents.
.1.3. ExtractionThe coarse powder of the air-dried rhizome was subjected to
uccessive solvent extraction using solvents of increasing polar-ty (petroleum ether, hexane, methanol, followed by ethanol). Theolvent was then distilled, evaporated and vacuum dried.
.1.4. Phytochemical AnalysisChemical tests were carried out on the various extracts of rhi-
ome of C. igneus (Ethanol, methanol, petroleum ether and hexane)sing standard procedures to identify the constituents as describedy Sofowara, Trease and Evans and Harborne [12–14]. The pres-nce or absence of different phytoconstituents viz. triterpenoidsSalkowski test), steroids, tannins, cardiac glycosides (Keller-Killaniest), saponin, phlobatannin, alkaloid and flavonoids, etc. weredentified in the extracts.
.1.5. High performance thin layer chromatographyuantification of flavonoids.1.5.1. Standard preparation. One milligram of quercetin and00 �g of kaempferol were dissolved in 1 ml of methanol in HPLCrade separately.
.1.5.2. High performance thin layer chromatography separationondition. Plant active constituents responsible for anti diabeticroperties were isolated by High Performance Thin Layer Chro-atography (HPTLC, Camag Muttenz, Switzerland). Acid hydrolysisas carried out on vacuum dried concentrated ethanol extract
f C. igneus rhizome to liberate aglycones, if any glycosides wereresent. The standards and sample solution were spotted on acti-ated HPTLC plates of silica gel F 254 of 0.5 mm thickness coatingsing a Camag Linomat V (Switzerland) sample applicator. Thelates (10 × 10) were developed with solvent system Toluene:Ethylcetate:Acetic acid:Methanol (2:7:0.25:0.25) to elute quercetinnd Toluene:Ethyl acetate:Methanol:Formic acid (6:3:0.2:0.4) foraempferol isolation. The developed plates were air dried andetected by 20% antimony chloride in chloroform, which was
prayed and dried in a chromatographic oven obtained at 105 ◦Cor 10 min. The plate was kept in Photo-documentation cham-er (CAMAG REPROSTAR 3) and the images were captured at UV54 nm. Finally, the plate was fixed in scanner stage and scanned at
entive Nutrition 3 (2013) 285–297
254 nm. The resolution bands were obtained and retardation factor(Rf) value were calculated.
2.1.5.3. Calibration curve of quercetin and kaempferol. A stock solu-tion of standard quercetin (1000 �g/ml) and standard kaempferol(100 �g/ml) were prepared in methanol. Different volume of stocksolution 1, 2, 3, 4, 5 and 6 �L, were spotted on TLC plate to obtain theconcentration of 1000, 2000, 3000, 4000, 5000 and 6000 ng/spot ofquercertin, respectively. For kaempferol different volume of stocksolution 1, 3, 5, 7, 9 and 12 �L, were spotted on TLC plate to obtainthe concentration of 100, 300, 500, 700, 900 and 1200 ng/spotof kaempferol respectively. The data of peak areas were plottedagainst the corresponding concentration.
2.1.5.3.1. Method validation.2.1.5.3.1.1. Precision. Methods of instrumental precision,
intra-assay precision, and intermediate precision were deter-mined. Instrumental precision was measured by replicate (n = 10)application of the same quercertin (concentration 2000 ng) andkaempferol standards solution (concentration 300 ng). Intra-assayprecision was evaluated by analysis of three replicate applicationsof freshly prepared standard solutions of same concentration, onthe same day. Intermediate precision was evaluated by analy-sis of three replicate applications of standard solution of sameconcentration on three different days.
2.1.5.3.1.2. Robustness of the method. By introducing smallchanges in the mobile phase composition, mobile phase volumeand duration of mobile phase saturation, the effects on the resultswere examined. Robustness of the method was done in triplicateat a concentration level of 4000 ng/spot for quecertin, 700 ng/spotfor kaempferol and the % of R.S.D peak area was calculated.
2.1.5.3.1.3. Ruggedness. The standard solution of concentra-tion 4000 ng/spot for quecertin, 700 ng/spot for kaempferol wereprepared and analyzed on 0, 6, 12, 24, 48 and 72 h. Data were treatedfor % R.S.D. to assess ruggedness of the method.
2.1.5.3.1.4. Limits of detection and Limit of quantification. Thelimits of detection (LOD) and the limits of quantification (LOQ) weredetermined as the amounts for which the signal-to-noise ratioswere 3:1 and 10:1, respectively.
2.1.5.3.1.5. Recovery studies. The accuracy of the method wasestablished by performing recovery experiments at three differ-ent levels using the standard addition method. In 2 �L (10 mg/mL)sample of ethanol extract of C. igneus, known amounts of que-certin (2000 and 3000 ng per spot) and kaempferol (300 and 500 ngper spot) standards were added by spiking. The values of percentrecovery and average value of percent recovery for quecertin andkaempferol were calculated.
2.1.5.3.1.6. Specificity. The specificity of the method was ascer-tained by analyzing the standard drug and extract. The spot forquercetin and kaempferol in the sample was confirmed by compar-ing the Rf values and spectra of the spot with that of the standard.The peak purity of the quercetin and kaempferol were assessed bycomparing the spectra at three different levels, viz. peak start, peakapex and peak end positions of the spot.
2.1.6. Isolation of flavonoid compoundCrude ethanol extract of C. igneus was chromagraphed on silica
gel column eluting with solvent mixtures increasing the polaritycomposed of chloroform, methanol (methanol to 35%) and frac-tions were collected. The purity of the all fractions collected wereanalyzed by thin layer chromatography on silica gel developedin Toluene:Ethyl acetate:Acetic acid:Methanol (2.5:7:0.25:0.25).Spots were visualized by spraying the plates with 20% antimony
chloride in chloroform, spray reagent. Fraction collected with n-hexane: ethyl acetate (increasing polarity methanol up to 35%)were pulled together where these fractions showed single spot ofsame Rf value of TLC. It was evaporated in a water bath (50–60 ◦C)
Prev
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P. Kalailingam et al. / Biomedicine &
o afford a solid residue. The residue was dissolved in a mixturef CHCl3: methanol (70:30) with little warming on a water bath. Itas left undisturbed in refrigerator when light yellowish powder
f flavonoid was obtained.
.1.6.1. Characterization of chemical compound. The purified com-ounds were analysed by GC-MS spectrometry in an LKB model091 at ionization energy of 70 eV. The 1H and 13C NMR spectraf the purified flavonoid compound were recorded at 300 MHz. Allhe 13C NMR spectra were recorded in the proton noise-decoupling
ode.
.2. Antidiabetic study
.2.1. AnimalsAlbino male rats of Wister strain (150–200 g) were used for the
tudy. The animals were maintained in an air-conditioned room forontroling temperature and humidity, where they were fed with atandard rat pellets feed supplied by Kamadhenu agencies, Banga-ore (India) and filtered water ad libitum. Animals in fasting wereeprived of food for > 16 hrs but allowed to access water freely.thical clearance, for the handling of experimental animals, wasbtained from Institute Animal Ethics Committee (265/CPCSEA).
.2.2. Acute toxicity evaluation in ratCiREE was tested for its acute toxicity (if any) in male rat. The test
as carried out by single oral administration of rhizome ethanolxtract at doses of 100, 200, 400, 600, 1200 and 2400 mg/kg to dif-erent groups of rats (five rats in each group). The mortality andeneral behaviors were observed continuously for one hour, 4 h,nd intermittently for next sixth hour and again at 24 h and 48 h15,16]. The parameters observed were gross behavioral changes,rooming, alertness, sedation, loss of righting reflex, tremors andonvulsions [17].
.2.3. Induction of diabetes mellitus in ratsDiabetes was induced by injecting STZ (Sigma, USA) at a dose
f 40 mg/kg bodyweight (bw) in 0.1 M cold citrate buffer of pH.5, interaperitoneally. STZ-injected animals exhibited severe gly-osuria and hyperglycemia and rats were stabilized over a periodf 7 days. Onset of diabetes was confirmed in the experimental ratsy measuring blood glucose concentration at 96 h after injection ofTZ. The rats with blood glucose level above 250 mg/dl were con-idered to be diabetic and were used for the experiment. Controlats were administrated with citrate buffer (pH 4.5) [18].
.2.4. The effect of single dose of CiREE on blood glucose levels inormal rats
This experiment involved testing of hypoglycemic effect of theormal rats after single oral administration. Group I served as aontrol; group II had the CiREE (100 mg/kg bw/day), group III hadiREE (200 mg/kg bw/day), blood glucose levels were determinedt 0, 30, 60, 90, 120 and 180 minutes.
.2.5. The effect of single dose of CiREE on blood glucose levels iniabetic rats
This experiment involved testing of hypoglycemic effect ofhe diabetic rats after single oral administration. Group I hadTZ-treated surviving diabetic rats; Group II had the STZ-induced
iabetic rats treated with CiREE (100 mg/kg bw/day), Group IIIad the STZ-induced diabetic rats treated with CiREE (200 mg/kgw/day). Blood glucose levels were determined at 0, 30, 60, 90, 120nd 180 minutes.
entive Nutrition 3 (2013) 285–297 287
2.2.6. Effect of CiREE on oral glucose tolerance in normal anddiabetic rats
After overnight fasting, a 0-min blood sample (0.2 ml) was takenfrom the rats in different groups viz., 6 groups of normal rats of6 rats/groups. Group I served as a control; Group II had CiREE(100 mg/kg bw/day), Group III had CiREE (200 mg/kg bw/day),Group IV had STZ-treated surviving diabetic rats; Group V had theSTZ-induced diabetic rats treated with CiREE (100 mg/kg bw/day),Group VI had the STZ-induced diabetic rats treated with CiREE(200 mg/kg bw/day), by oral administration, without delay a glu-cose solution (2 g/ml per kg) was administered by gavage. Six moresamples were taken at 30, 60, 90,120,150 and 180 min after glucoseadministratio1 [19].
2.2.7. Experimental designAnimals were divided into five groups with six animals each.
Group I served as a control; group II had STZ-treated surviving dia-betic rats; group III had the STZ-induced diabetic rats treated withCiREE (100 mg/kg bw/day), group IV had the STZ-induced diabeticrats treated with CiREE (200 mg/kg bw/day), group V served as apositive control and received glibenclamide (0.5 mg/kg/bw) for 30day, orally administrated. Rats were fasted and sacrificed at the ter-mination of the experiment i.e. on the 30th day the blood sampleswere collected to analyze the effect of C. igneus rhizome extract onbiochemical parameters.
2.2.7.1. Collection of pancreas and Blood. At the end of treatment,blood was collected by cardiac puncture and serum was separatedby centrifugation at 2500 rpm. The rats were sacrificed by cervicaldislocation and pancreas was excised immediately and thoroughlywashed with ice-cold physiological saline. The serum collected wasused for biochemical estimation.
2.2.7.2. Estimation of biochemical parameters. Glucose level wasobserved by onetouch horizone (Lifescan, Milipitas, CA, USA) Glu-cometer in heparinised tubes, HbA1C [20] was estimated accordingto the method of Sudhakar Nayak and Pattabiraman and Seruminsulin (Boerhringer Mannheim kit, Germany) levels were ana-lyzed.
2.2.7.3. Estimation of glycogen in liver. This estimation indicatesthe distinction between ‘free’ and fixed glycogen content in tissueby using anthrone reagent (solution containing anthrone-0.05%,thiourea-1% and sulphuric acid-72%, potassium hydroxide-30%,ethanol-95%). Accurately weighed 100 mg of liver was digestedwith 2 ml of 30% boiling KOH, 3 ml of 95% ethanol was added andheated until the bubbles was formed. These mixtures was cooledand centrifuged at 1000 rpm for 5 min and supernatant was dis-carded. The residue was dissolved in 2 ml of distilled water, 10 ml ofanthrone reagent was added and immersed in an ice bath to preventexcessive heating. Tubes were incubated at 100 ◦C for 4 minutes forcolor development and immersed in an ice bath. Absorbance wasmeasured at � 620 nm using a spectrophotometer. The glycogencontent in wet tissue was expressed as mg of glycogen per 100gm[21].
2.2.7.4. Determination of effect of CiREE on lipid profile of diabetic rats.The total cholesterol and triglyceride levels of each serum sam-ple were separately determined by enzymatic colorimetric methodusing reagent kits [22]. Lipid levels of diabetic animals were mea-sured before (basal) and after the induction of diabetes as well as on15th and 30th day after commencement of treatment. The choles-
terol and triglyceride levels were determined using commerciallyavailable reagent kits (QCA, South Africa) following the manufac-turer’s instructions. The absorbance of each sample containing thereaction mixtures with or without serum was read at 540 nm in a
288 P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297
Table 1Qualitative phytochemical analysis in different extracts of Costus igneus rhizome.
S. No Extracts Tannin Phlobatannin Saponin Flavonoid Steroid Terpenoids Cardiac glycosides
1 Ethanol + + + + + + +
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3.2.3. AccuracyAll the samples of various formulations were spiked with
the known amount of standard, and the percent ratios betweenthe recovered and expected concentrations were calculated.
Table 2Method validation data for HPTLC quantification of quercetin and kaempferol inethanol extract of Costus igneus rhizome.
8 Intra-day variation (RSD [%]n = 3) on the same day
0.30 0.31
2 Methanol + – +
3 Hexane – – +
4 Petroleum Ether – – +
V spectrophotometer (Shimadzu-1601, Japan). Total cholesterolr triglyceride was calculated using the formula: Total cholesterolmg/dl) = SAO.D/STO.D × 200, where SAO.D = optical density of testample and STO.D = optical density of standard.
Low density lipids (LDL) and high density lipids (HDL) wereetermined by standard procedures in an auto analyzer usingcolin kits (E. Merck, Mumbai, India).
.2.7.5. Histopathological Investigation. Pancreas was washed withaline and a small tail portion of this organ was fixed in 10% forma-in. Then the tissues were processed by standard histopathologicalechnique (i.e. dehydration through graded isopropyl alcohol,leaning through xylene impregnated in paraffin wax for 2 h).
ax blocks were made. Sections were made using microtome andtained by haematoxylin eosin method and photographed.
.2.7.6. Electron microscopical (EM) studies of pancreas. Small frag-ent of pancrease was fixed with 3% glutaraldehyde in sodium
hosphate buffer (200 mM, pH 7.2) for 3 h at 4 ◦C. Materials wereashed with same buffer and post fixed in 1% osmium tetroxide and
n sodium phosphate buffer (pH 7.4) for 1 h at 4 ◦C. Tissue samplesere washed with same buffer for 3 h at 4 ◦C and were embedded
n araldite. Sixty to 90 nm sections were prepared with LKBUM4ltramicrotome using a diamond knife and sections were mountedn a copper grid and stained with uranyl acetate and reynolds leaditrate. The grids were examined under a Phillips Electron Micro-cope model (EM201 C).
.2.8. Statistical analysisStatistical analysis was performed using the Origin lab, version
.5. The values were analyzed by One-way Anova, Means Com-arison using Bonferroni Test. All the results were expressed asean ± SD for six rats in each group and P < 0.05 was considered as
tatistically significant.
. Results
.1. Phytochemical analysis
Compounds of different polarity from dried rhizome powder of. igneus were extracted using different solvents such as petroleumther, hexane, chloroform, ethanol and water. These extracts wereubjected to preliminary phytochemical screening for the presencef different chemical group. Of all extracts tested, ethanol extractas found to contain the highest number of phytochemicals such
s tannins, phlobatanin, saponin, flavonoids, steroid, terpenoids,ardiac glycosides (Table 1). Based on this results ethanol extractas used for further study of antidiabetic compounds isolation
uch as naturally occurring phenolic compounds such as querce-ine (Rf 0.72) and kaempferol (Rf 0.35) were isolated by HPTLConformed to standard quercetine (Rf 0.72) (Fig. 1) and kaempferol0.35) (Fig. 2) respectively.
+ – + +– + – –– + + –
3.2. High performance thin layer chromatography quantificationof quercetine and kaempferol
Different compositions of solvents were used for HPTLC anal-ysis in order to obtain high resolution and reproducible peaks.The desired aim was achieved using Toluene:Ethyl acetate:Aceticacid:Methanol (2:7:0.25:0.25) for quercetine, and Toluene:Ethylacetate:Methanol:Formic acid (6:3:0.2:0.4) for kaempferol as themobile phase. The wavelength of 254 nm (After derivatization –366 nm) was found to be optimal for highest sensitivity which gavethe single peak at Rf 0.72 for quecertin and at Rf 0.35 for kaempferolEthanol extract of C. igneus rhizome consist of quecertin (0.794%)(Fig. 1) and kaempferol (4.2%) (Fig. 2) in its composition of con-stituents (Table 2).
3.2.1. Linearity, limit of detection and limit of quantificationUnder the above described experimental conditions, linear cor-
relation between the peak area and applied concentration wasfound to occur in the concentration range of 1000 to 6000 ng/spot ofstandard quecertin (Fig. 1(e)) and 100 to 1200 ng/spot of standardkaempferol (Fig. 2(e)). The correlation coefficient of quecertinand kaempferol were found to be 0.99677 and 0.99935 respec-tively. The peak area (y) is proportional to the concentration ofquecertin and kaempferol (x) following the regression equationy = 1625.724 +4.792x and 38.608 + 4.815x respectively. The exper-imentally derived LOD (quercertin-4 ng and kaempferol-5 ng andLOQ (quercertin-99 ng and kaempferol-99.4 ng) were determined.
3.2.2. PrecisionPrecision data on repeatability (intra-day) and instrumental
variation for three different concentration levels were studied. Bothprecision studies showed R.S.D. less than 1%, indicating a sufficientprecision.
9 Inter day variation (RSD [%]n = 3) on three successive days
0.22 0.24
10 Accuracy [%] 96.98 93.511 Specificity Specific Specific
P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297 289
Fig. 1. (a) Structure of Quercetin, (b) HPTLC plate for Quercetin isolation, curve, (c) HPTLC chromatogram of standard Quercetin, (d) HPTLC chromatogram for isolation ofQ
Skmk
3
i(M
3.2.5. Ruggedness
uercetin in rhizome, (e) linearity curve.
atisfactory recoveries of 96.98% of quercertin and 93.5% ofaempferol indicate that the proposed simultaneous HPTLCethod is reliable for the quantification of quercertin and
aempferol.
.2.4. Robustness
The mobile phase composition was altered by ± 2% changes
n the ratio of Toluene:Ethyl acetate:Acetic acid:Methanol2:7:0.25:0.25) to elute quercetin and Toluene:Ethyl acetate:
ethanol:Formic acid (6:3:0.2:0.4) for kaempferol isolation and
also in detection of wavelength (254 nm, after derivatization366 nm). No changes were observed in retention time and peakshape.
Low % R.S.D. value of 0.28 between the peak area values provedthe ruggedness of the method indicating that quercetine, andkaempferol is stable during the extraction procedure as well asduring analysis.
290 P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297
F PTLCk
3
bpok
3c
e
ig. 2. (a) Structure of kaempferol, (b) HPTLC plate for Kaempferol isolation, (c) Haempferol in rhizome and leaf respectively, (e) linearity curve.
.2.6. SpecificityThe peak purity of quercetine, and kaempferol were assessed
y comparing the spectra at peak start, peak apex and peak endositions of the spot. The correlation (r = 0.99677, r = 0.99935) werebtained between the standard and the sample of quercetine andaempferol respectively.
.3. Isolation, purification and characterization of flavonoid
ompound
Flavonoid compound was isolated and purified from ethanolxtract of C. igneus rhizome by repeated silica gel column
chromatogram of standard kaempferol, (d) HPTLC chromatogram for isolation of
chromatography followed by preparative TLC (Rf 0.72). 1H NMRspectrum of isolated flavonoid derivative is 6.24, 6.4 (Phenolic car-bons attached with flavones ring), 6.8, 7.8, 7.9 (side chain phenylring), 3.5 (methoxy), 1.0 to 2.5 (alcohol protons) (Fig. 3 (a), (b)). 13CSpectrum of isolated quecertin derivative is 163, 161, 147, 144, 146,135, 114 (Phenolic carbons attached with flavones ring), 177 (car-bonyl carbon), 122, 120, 114, 97 (side chain phenyl ring) (Fig. 3(c)
(d)).
The mass spectrum of purified compound contained signalsof m/z 995.38 (MI), 398, 355, 342, 327, 300, 282, 271 and126.
P. Kalailingam et al. / Biomedicine & Prev
Fig. 3. (a) H1 NMR spectrum of quercetin derivative, (b) Enlarge of H1 NMR spec-trum, (c) 13C NMR spectrum of quercetin derivative, (d) Structure of quercetinderivative.
entive Nutrition 3 (2013) 285–297 291
3.4. Acute toxicity studies
In the present study, toxicity test was carried upto high con-centration of 2400 mg/kg (12 times more than chosen dose). Evenat this dose, extract did not exhibit any sign of toxicity. Since themain purpose of this test is to get some idea on conspicuous behav-ioral changes and death, if any but CiREE did not exhibit any toxicsymptoms in the limited toxicity evaluation in rat.
3.5. Single dose and glucose tolerance study
Administration of single dose of 100 or 200 mg/kg body weightwas found to reduce blood glucose level in normal rats. The max-imum reduction in serum glucose level was noted at 2.5 hrs by200 mg/kg per bw (25%) followed by 100 mg/kg per bw (20%)(Fig. 4(a)). The effect of plant extract in STZ-induced diabetic rats isshown in Fig. 4(b). The fasting blood glucose levels in STZ-induceddiabetic rats were reached above 280 mg/dl. During the single dosestudy, the results were found to be similar to that of normal ani-mals. The maximum effect was seen at 2.5 hrs by 200 mg/kg per bw(64%) followed by 100 mg/kg per bw (43%). There was tendency toincrease serum glucose after 3.0 hrs.
Fig. 5(a) and (b) depict the effects of the above mentioneddose of CiREE on glucose tolerance up to 3 h in normal and dia-betic rats. The 57%, 47%, 27%, 56%, 34%, 20% of increased bloodglucose levels were observed within 1hr after oral administrationof glucose in Group I to VI respectively. Doses of 200 mg/kg/bw.caused a significant decrease in the blood glucose levels after90 min in the glucose pulse; 100 mg/kg/bw led to a slight reduc-tion in glycemia compared to the strong effect above. These factsdemonstrate a dose-dependent activity for CiREE in the glucosetolerance test. The maximum reduction was seen within 2.5 hby CiREE (200 mg/kg/bw) followed by CiREE (100 mg/kg/bw) at3.0 hrs.
3.6. Biochemical parameter
Table 3 reveals the comparison of glucose concentration inanimals of control group, animals received STZ against the ani-mals received CiREE at the dose of 100 mg/kg and 200 mg/kg andstandard drug i.e. glibenclamide in a dose of 0.5 mg/kg at 0th,15th and 30th days. The glucose level was significantly (P < 0.05)higher in diabetic control (group II) compared with normal con-trol (group I). On other hand, the level of glucose was significantly(P < 0.05) decreased in group III, IV at 15th and 30th day, simi-lar results were also found in glibenclamide treated diabetic rats(group V). There was no significance (P < 0.05) difference betweenCiREE 100 mg/kg (group III) and glibenclamide (group V), whichindicates that 100 mg of CiREE has more or less equal efficacy toreference drug glibenclamide.
Table 4 showed that the levels of liver glycogen, insulin andHbA1c were studied in normal control, diabetic control, CiREE(100 mg/kg and 200 mg/kg) and glibenclamide treated STZ-induceddiabetic rats. The liver glycogen and serum insulin were found to besignificantly decreased (P < 0.05) in diabetic control (group II) com-pared with normal control (group I) whereas the level of HbA1cwas significantly (P < 0.05) increased in diabetic control (Group II)when compared to normal control. Oral administration of CiREE(group III and IV), and glibenclamide (group V) for 30 days resultedthat reverse back to normal levels. The results showed that the dose200 mg/kg was more effective than 100 mg/kg of CiREE.
The lipid profile in control and experimental rats are depicted in
Table 5. In STZ-induced diabetic rats (group II), there was a signifi-cant (P < 0.05) increase of total cholesterol, triglycerides, LDL, VLDLand significant (P < 0.05) decrease in HDL cholesterol in serum com-pared with normal control (group I). Oral administration of CiREE
292 P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297
Fig. 4. (a) Effect of single dose of CiREE on Glucose level in normal rats, (b) Effect of single dose of CiREE on Glucose level in STZ-induced diabetic rats.
Fig. 5. (a) Effect of CiREE on GTT in normal rats, (b) Effect of CiREE on GTT in STZ-induced diabetic rats.
Table 3Effect of CiREE on blood glucose at 0th, 15th and 30th day.
S. No Groups 0th day (mg/dl) 15th day (mg/dl) 30th day (mg/dl)
1 Group I (Normal control) 91.75 ± 0.647 91.225 ± 0.759 91.55 ± 1.2692 Group II (Diabetic control) 284.675 ± 1.299a 285.13 ± 1.315a 285.13 ± 1.315a
Values represent mean ± SD (n = 6); comparisons between groups are as follows a: group I vs group II, b: group II vs groups III, IV, V, c: group III vs groups IV, V. Statisticalsignificance is as follows:
a P < 0.05.b P < 0.05.c P < 0.05 (0th, 15th and 30th day values are compared with other 0th, 15th and 30th day values respectively).
P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297 293
Table 4Effect of CiREE on liver glycogen, Insulin, HbA1c at 30th day.
S. No Groups Day Liver glycogen Insulin HbA1c
1 Group I (Normal control) 0th day 10.38 ± 0.63 15.2 ± 0.71 5.32 ± 0.10215th day 11.2 ± 0.57 14.9 ± 0.45 5.2 ± 0.2730th day 10.808 ± 0.165 15.57 ± 0.513 5.38 ± 0.35
2 Group II (Diabetic control) 0th day 4.92 ± 0.375a 5.9 ± 0.27a 7.4 ± 0.253a
Values represent mean ± SD (n = 6); comparisons between groups are as follows a: group I vs group II; b: group II vs groups III, IV, V; c: group III vs groups IV, V. Statisticalsignificance is as follows:
a P < 0.05.b P < 0.05.c P < 0.05 (0th, 15th and 30th day values are compared with other 0th, 15th and 30th d
Expression units.
Parameters Unit
Liver glycogen mg/g of wet tissue
(vcctd
TE
Vs
Insulin �IU/mlHbA1c mg/g of HB
group III, IV) and glabenclamide for 30th days lowered the highalues back to normal levels. The levels of total cholesterol, trigly-
erides, LDL, VLDL were significantly (P < 0.05) decreased and HDLholesterol was significantly (P < 0.05) increased. This indicates thathe rhizome extracts had favorable effects on lipid metabolism ofiabetic rats.
able 5ffect of CiREE on lipid profile at 0th, 15th and 30th day.
Groups Treatment days TC (mg
Group I (Normal Control) Basal 810th day 82.115th day 82.530th day 82.13
Group II (Diabetic Control) Basal (Before diabetic induction) 80.80th day (After diabetic induction) 123.615th day 122.4630th day 122.3
Group III (CiREE 100 mg/kg) Basal (Before diabetic induction) 79.30thday (After diabetic induction) 123.315th day 106.830th day 90.2
Group IV (CiREE 200 mg/kg) Basal (Before diabetic induction) 80.30th day (After diabetic induction) 123.315th day 91.730th day 83.2
Group V (Glibenclamide) Basal (Before diabetic induction) 80.10th day (After diabetic induction) 120.115th day 106.230th day 91.9
alues represent mean ± SD (n = 6); comparisons between groups are as follows a: groupignificance is as follows:
a P < 0.05.b P < 0.05.c P < 0.05 (0th, 15th and 30th day values are compared with other 0th, 15th and 30th d
ay values respectively).
3.7. Histopathological investigation
Histopathology of the pancreas in control animals showednormal pancreatic parenchymal cell and islet (Fig. 6(a)). In dia-betic control, pancreas showed moderate hyperplasia of islet cells,more number of voculation, and severe congestion in pancreaticparenchyma, number of islet was reduced and mild infiltrationof inflammatory cells (Fig. 6(b)). In diabetic animals treated withethanolic rhizome extract, pancreas section showed hyperplasiaof islet was recovered, mild congestion of pancreatic parenchyma
and number of islet increased (Fig. 6(c) and (d)). In diabetic animaltreated with glibenclamide showed increased number of pancreaticislet (Fig. 6(e)).
I vs group II; b: group II vs groups III, IV, V; c: group III vs groups IV, V. Statistical
ay values respectively).
294 P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297
F duced( (200
3
scemntNbohmmt(icr
4
tdtozsmra
eomt
ig. 6. (a) H & E stained pancreas of normal rat, (b) H & E stained pancreas of STZ-in100 mg/kg), (d) H & E stained pancreas of diabetic rat treated with rhizome extract
.8. Electron microscopic studies
�-cells of STZ-induced diabetic rats at the 30th day pos-essed rounded nuclei with a moderately distinct nucleolus. Theytoplasm of the cells moderately varied of its dense. Roughndoplasmic reticulum was mostly seen in the cytoplasm. Theitochondria are medium-sized, oval, and possess a moderate
umber of mainly transverse cristae. Free ribosomes, hyper-rophied golgi complex, were also observed in the cytoplasm.ormal control rats pancreas, �-cells contained increased num-er of mature secretory granules. The granules were composedf a central core, usually of moderate homogeneous, or slightlyeterogeneous electron density, and an external single-layeredembrane with a rather large space between the core and theembrane (Fig. 7(b)). The granules were diffusely distributed in
he cytoplasm. On 30th day of diabetic rats treated with CiREE200 mg/kg/bw) �-cell contained remarkably increased number ofmmature secretory granules, mitochondria and of hypertrophiedytoplasmic organelles such as golgi complex and endoplasmiceticulum (Fig. 7(c) and (e)).
. Discussion
DM is a serious chronic metabolic disoder. Effective control ofhe blood glucose level is a key-step in preventing or reversingiabetic complications and improving the quality of life in bothypes 1 and 2 diabetic patients [23–25]. The chemical constituentsf C. igneus was studied showed that the ethanol extract of rhi-ome consist of terpenoids, steroids, tannins, cardiac glycosides,aponin, phlobatannin, alkaloid and flavonoids while compared toethanol, hexane and petroleum ether extract. Based on these
esults we have chosen the ethanol rhizome extracts for furtherntidiabetic compounds analysis and antidiabetic activity study.
Antihyperglycemic potency of the C. igneus rhizome ethanol
xtract (CiREE) in diabetic rat has been indicated here by the studyf fasting blood glucose levels, as the important basal parameter foronitoring of diabetes and the dosage of 200 mg/kg is more effec-
ive than that of 100 mg/kg. The increased level of glycosylated Hb
diabetic rat, (c) H & E stained pancreas of diabetic rat treated with rhizome extractmg/kg), (e) H & E stained pancreas of diabetic rat treated with glibenclamide.
observed in STZ-induced diabetic rats might be due to decreasedformation of Hb. It has been reported that in diabetic mellitus,the total haemoglobin level is much lower than the normal leveland increased levels of glycosylated Hb. Earlier report states thatduring DM, excess of blood glucose reacts with hemoglobin lead-ing to the formation of HbA1c[26]. The level of HbA1c is alwaysmonitored as a reliable index of glycemic control in diabetes. Ele-vated levels of HbA1c and reduced levels of Hb observed in ourstudy reveal that diabetic animals had prior high blood glucoselevel. Administration of CiREE (100, 200 mg/kg bw/day) has low-ered the elevated HbA1c levels to near normal level. It has alreadybeen reported that decreased liver glycogen level is due to insulindeficiency and associated glycogenolysis [27]. The possibility ofrestoring liver glycogen level in STZ-induced diabetic rats by theadministration of CiREE may be due to increased insulin secretionand reactivation of glycogen synthase enzyme system. Severe DMis associated with hyperlipidemia [28]. The levels of TC and TGhave been decreased significantly in diabetic rat after CiREE supple-mentation. These effects may be due to low activity of cholesterolbiosynthesis enzymes and or low level of lipolysis, which are underthe control of insulin [29]. The CiREE supplementation also resultsto the significant attenuation in the level of serum HDL towardsthe control level, which again strengthens the hypolipidemic effectof this extract. There are reports that other medicinal plants havehypoglycemic and hypolipidemic effects that could prevent orbe helpful in reducing the complications of lipid profile seen insome cases of diabetes in which hyperglycemia and hypercholes-terolemia coexist [29].
Pancreatic lesions induced by STZ leads to full blown diabeteswhich were reversed upon by the treatment with CiREE whichpossibly stimulate the regeneration of pancreatic islets cell. STZis known to induce chemical diabetes by selective destruction ofpancreatic �-cells through three processes viz: DNA nitric oxideproduction, alkylation and free radical generation [30]. This was
observed in this study that diabetic control rat pancreas showedmarkedly reduced and sunken islets mass, infiltrated by lympho-cytes – general fibrosis. This was observed from various reportsin literature of STZ damage to pancreas [31,32] and also another
P. Kalailingam et al. / Biomedicine & Preventive Nutrition 3 (2013) 285–297 295
F ) Panc( as of d
dmTngwtp
plmafrt
wt
ig. 7. Electron microscopical studies of pancreas. (a) Pancreas of normal rat, (b100 mg/kg), (d) Pancreas of diabetic rat treated with CiREE (200 mg/kg), (e) Pancre
iabetogenic agent alloxan [33]. As with these cited works, treat-ent with our extracts had ameliorated and reversed the lesions.
he CiREE with the ability to ameliorate this type of diabetes willecessarily address in part or whole, the reactive oxygen specieseneration process. This implies that the plant must be endowedith antioxidants compounds which would be reverse the cyto-
oxic cycle of STZ in the pancreas, or at least mop up or ‘quench’ theopulation of ROS in circulation.
Earlier report from our laboratory has also demonstrated theresence of phytochemicals with known antioxidant properties
ike the flavonoids, polyphenols and terphenoids (antioxidant vita-ins and micronutrients) in the leaves of these plants [11]. The
ction of these phytochemicals and nutrients may have arrested theree radical generation process or mop up the circulating radicalsesponsible for histological lesions and complications of diabetes,
hereby allowing for regeneration of pancreatic islets cells.
In present study quercetin, quercetin derivative and Kaempferolere isolated as a antidiabetic compounds of CiREE, it might con-
ribute to the reduction in blood glucose level and also lipid profile
reas of STZ-induced diabetic rat, (c) Pancreas of diabetic rat treated with CiREEiabetic rat treated with glibenclamide.
in STZ-induced diabetic rats. This correlates with the previousreports [34–36] that diosgenin possess hypoglycemic property. Theeffect of quercetin on decreasing the level of cholesterol VLDL andLDL were also determined by Nuraliev and Avezov [37]. Regener-ation of the islets of langerhans by quercetin is in agreement withthe data of Chakravarthy et al. [38], who specifically demonstratedthe regeneration and functional activity of regenerated �-cells byflavonoids. The ability of quercetin in significantly increasing theglucokinase activity of the liver and reducing plasma cholesterolTG in diabetic animals could be explained by the work of Hii andHowell [39]. Many earlier reports revealed that the kaempferolderivatives containing plant extracts has hypoglycemic activitysuch as kaempferol-3-O-galactoside and Kaempferol-3-rhamno-O-glucoside from Bahvina variegate [40], Kaempferol-3-rhamnosidefrom Zizhyphus rugosa [41], Kaempferol-3-O-glucopyranoside from
Morus insignis [42], Kaempferol-3-O-(2gal-rhamnosilobonoside)from Sterculia rupestris [43] and Kaempferol-3-O-isophoroside-4-O-beta-D-glucoside is one of the responsible factors for thehypoglycemic effect of E. myriochactum.
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96 P. Kalailingam et al. / Biomedicine &
Glibenclamide is used as a standard drug to compare the activ-ty of CiREE in a dose of 100 mg/kg and 200 mg/kg. The comparableffect of the extract (200 mg/kg) with glibenclamide (0.5 mg/kg)ay suggest similar mode of action, since the mechanism of action
f glibenclamide is the stimulation of insulin release and the inhibi-ion of glucagon secretion. It has been described that glibenclamides effective in moderate diabetic state and ineffective in severeiabetic animals where pancreatic �-cells are totally destroyed44,45]. The hypoglycaemic effect of medicinal plant extracts isenerally depends upon the degree of �-cell destruction [46].he possible mechanism by which plant extract brings about itsypoglycemic action may be by potentiating the insulin effect by
ncreasing either the pancreatic secretion of insulin from �-cells47]. The findings also suggest that plant extracts may regenerate
cells and has protective effect on �-cells from glucose and lipidoxicity. As other studies showed, plant extracts might bring aboutts hypoglycemic effect through insulin secretion from the � cellsf ilets and insulin sensitivity [48,49].
EM studies revealed that the number of mature secretory gran-les and hypertrophied golgi complexes were increased at 30thay of diabetic rats treated with CiREE (200 mg/kg bw/day). Ham-guchi et al. (1986) suggested that the ultrastructure of the islet cane affected by various glucose and oxygen concentration. Firstly,yperglycemia (in diabetic rats) with high oxygen induced theypertrophy of endoplasmic reticulum and golgi complex, an abun-ance of free ribosomes, and degranulation and the margination ofecretory granules in the �-cells. In the present study, abundantmmature secretory granules and partially dilated endoplasmiceticulum and golgi complexes and increased mitochondria werebserved at the 30th day of diabetic rat treated with CiREE200 mg/kg bw/day).
The possible mechanism by which CiREE brings about its hypo-lycaemic action may be due to flavonoids present in this planthich stimulates the receptor on the cytoplasm side of the mem-
rane, a protein phosphokinase of the tyrosine-specific type. Ithosphorylates itself with the aid of ATP, undergoes a confor-ational change, and activates via a G-proteins, which liberates
everal second messenger to activate protein P-kinases which opena2+ influx gives insulin like effect [50]. Another possible mecha-ism may be due to alkaloids and sapogenin causes inhibition ofitochondrial function that increases the AMP/ATP ratio, which
ould explain the activation pathway in the treatment of dia-etes [51]. The third most important probable mechanism of actionay be by potentiating the insulin effects of plasma by increas-
ng either the pancreatic secretion of insulin from the existing-cells or by its release from the bound form. Though the exactechanism is unknown but we are assuming that various active
onstituents of this plant help to improve in treatment of dia-etes.
In conclusion the data obtained from the present study indicateshat the C. igneus may have beneficial effects as both antidia-etic, antihyperglycemic agents and also warrants further studieso isolate and characterize potent molecules for DM and its lipidsssociated complications.
. Acknowledegments
Dr. T. Eevera acknowledges DBT, New Delhi (BT/PR10018/NNT/8/95/2007) for providing financial support for this project.
isclosure of interest
The authors declare that they have no conflicts of interest con-erning this article.
[
entive Nutrition 3 (2013) 285–297
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